Dong-Jun Seo

Antifungal activity of gallic acid purified from Terminalia nigrovenulosa bark against Fusarium solani.

The antifungal activities of methanolic extracts from Terminalia nigrovenulosa bark (TNB) was investigated for effects on the initial growth of mycelia against Fusarium solani. The ethyl acetate fraction separated from TNB demonstrated the highest antifungal activity against F. solani. The antifungal compound was isolated from TNB using silica gel column and Sephadex LH-20 chromatography combined with thin-layer chromatography and high performance liquid chromatography. Structural identification of the antifungal compound was conducted using (1)H NMR, (13)C NMR, and liquid chromatography-tandem mass spectrometry. The purified antifungal compound was gallic acid (GA) or 3,4,5-trihydroxy benzoic acid. Purified-GA possesses the high antifungal activity against F. solani, and that antifungal activity was dosage-dependent. The hyphae became collapsed and shrunken after 24 h incubation with GA (500 ppm). In pot experiments, the application of TNB crude extract was found to be effective in controlling the cucumber Fusarium root rot disease by enhancing activities of chitinase, peroxidase thereby promoting the growth of plants. The applied TNB extract significantly suppressed root rot disease compared to control. It resulted in 33, 75 and 81% disease suppression with 100, 500 and 1000 ppm of TNB crude extract, respectively. The study effectively demonstrated biological activities of the TNB extract, therefore suggesting the application of TNB for the control of soil-borne diseases of cucumber plants.

Antimycotic activities of Cinnamon-derived compounds against Rhizoctonia solani in vitro

In this study, the effects of medicinal plant extracts on the development of mycelium in the following phytopathogenic fungi were evaluated: Phytophthora capsici, Rhizoctonia solani, Fusarium solani, Colletotrichum gloeosprorioides, and Botrytis cinera. Of the 26 medicinal plants tested, six plant extracts showed antifungal activity against phytopathogenic fungi. The highest antifungal activity was exerted against R. solani by the n-hexane fraction of a Cinnamon (Cinnamomum cassia Blume) solvent extract. Therefore, the antifungal compound fractions I and II were purified from the n-hexane fraction by TLC on silica gel plates. When treated with solutions containing compound fractions I or II at a concentration of 2%, the mycelia growth rate of R. solani was reduced to 0.19 and 0.18, respectively. In addition, microscopic observation of the hyphal morphology of R. solani following treatment with compound fraction I revealed the presence of severely damaged hyphae. Specifically, the hyphal tips became swollen, collapsed or were completely destroyed in response to treatment with solution containing compound fraction I at concentration of 1%.

Nematicidal activity of 3,4-dihydroxybenzoic acid purified from Terminalia nigrovenulosa bark against Meloidogyne incognita ☆

In this study, the 3,4-dihydroxybenzoic acid (3,4-DHBA) from Terminalia nigrovenulosa bark (TNB) was purified and its in vitro nematicidal activity was investigated against Meloidogyne incognita. The purification of 3,4-DHBA used a silica gel column and Sephadex LH-20 chromatography combined with thin-layer chromatography and high performance liquid chromatography. Structural identification of the 3,4-DHBA was conducted using (1)H nuclear magnetic resonance (NMR), (13)C NMR, and liquid chromatography time-of-flight mass spectrometry. Nematicidal activity bioassays revealed that 3,4-DHBA treatment resulted in 33.3, 47.5, 72.5 and 94.2% J2 mortality at 0.125, 0.25, 0.5 and 1.0 mg/ml, respectively after 12 h incubation. J2 mortality was increased significantly (P < 0.0001) with increasing incubation time in the range of 54.2-94.2% from 3 to 9 h after incubation with 3,4-DHBA (1.0 mg/ml), but with no significant difference observed where the incubation time was increased from 9 to 12 h. The 3,4-DHBA treatment resulted in 33.3, 65.0, 76.7 and 85.0% hatch inhibition at 0.125, 0.25, 0.5 and 1.0 mg/ml, respectively, 3 days after incubation. Changes in the shape of the eggs were determined after incubation for 1 day with a 3,4-DHBA concentration of 1.0 mg/ml.

Nematicidal activity of compounds extracted from medicinal plants against the pine wood nematode Bursaphelenchus xylophilus

To investigate in vitro nematicidal activity against the pine wood nematode, Bursaphelenchus xylophilus, plant methanolic extracts were obtained from 26 medicinal plants and herbs in Vietnam. Of the plant extracts tested, a 10 mg ml−1 concentration of Cinnamomum cassia bark extract showed the highest level of nematicidal activity against juvenile and adult B. xylophilus after treatment for 7 h. The remainder of the plant extracts had little or no effect on juveniles or adults. Four solvents, n-hexane, chloroform, ethyl acetate and n-butanol, were used initially to partition the compounds obtained from C. cassia extracts. The compound (5 mg ml−1) in the n-hexane fraction showed the highest nematicidal activity at 60 min after treatment. Compounds III and IV were then separated from the n-hexane fraction using silica gel plates. These compounds showed very similar activity against the pine wood nematode. However, treatment with different concentrations of these compounds resulted in a significant difference in their effects on the mortality of the pine wood nematode, with greater concentrations leading to increased mortality after the same exposure time. Overall, treatment with 3 mg ml−1 of compounds III and IV resulted in greater than 95% mortality against juvenile and adult B. xylophilus at 50 min after treatment.

Nematicidal activity of gallic acid purified from Terminalia nigrovenulosa bark against the root-knot nematode Meloidogyne incognita

The nematicidal activity of Terminalia nigrovenulosa bark (TNB) and its purified compound were assayed against Meloidogyne incognita in vitro. The nematicidal compound was isolated from TNB using silica gel column and Sephadex LH-20 chromatography combined with thin-layer chromatography and high performance liquid chromatography. Structural identification of the nematicidal compound was conducted using 1H-nuclear magnetic resonance (NMR), 13C-NMR and liquid chromatography-tandem mass spectrometry. We found that the nematicidal compound purified from TNB was gallic acid (GA) or 3,4,5-trihydroxy benzoic acid. Nematicidal activity bioassays revealed that GA treatment resulted in 20.3, 37.5, 73.3, 88.3 and 95.8% hatch inhibition at 0, 0.25, 0.5, 1.0 and 2.0 mg ml−1 after 3 days, respectively, of incubation. Eggshells appeared to be deformed and destroyed at 2 and 3 days after incubation with a GA concentration of 1.0 mg ml−1, respectively. Additionally, after treatment with a GA concentration of 1.0 mg ml−1, mortality of second-stage juveniles of M. incognita was 65.0, 75.0, 96.7 and 100% at 3, 6, 9 and 12 h incubation, respectively.

Expression patterns of chitinase produced from Paenibacillus chitinolyticus with different two culture media

To investigate the expression patterns of chitinase isozymes on native-PAGE and SDS-PAGE gels Paenibacillus chitinolyticus MP-306 was cultured on culture media with and without chitin substrate. P. chitinolyticus MP-306 had a strong chitinolytic activity on colloidal chitin medium. Chitinase isozymes of MP-306 were expressed as six bands (CN1-CN6) on native-PAGE gels and thirteen bands (CS1-CS13) on SDS-PAGE gels after incubation in chitin medium. Three bands (CN1, CN2, and CN3) of chitinase isozymes of MP-306 on native-PAGE gels were expressed as nine bands (CS1, CS2, CS3, CS4, CS5, CS6, CS8, CS10, and CS13) of chitinase isozymes on SDS-PAGE gels. Three bands (CN4, CN5, and CN6) of chitinase isozymes of MP-306 were strongly inhibited by metal ions on native-PAGE and SDS-PAGE gels.

Detection of chitinase ChiA produced by Serratia marcescens PRC-5, using anti-PrGV-chitinase

In this study, a bacterium Serratia marcescens PRC-5 that displayed strong chitinolytic activity on 0.5% colloidal chitin-containing agar medium was isolated from soil. The chitinase activity increased rapidly with a maximum level (6.14 U/mL) on 4 days of incubation with swollen chitin (pH 5.0). Three active bands of chitinase isozymes were observed (53, 44, and 34 kDa) on SDS-PAGE gel and there pI values ranged from pI 5.4 to 5.8 on 2D gels. The chitinase from the PRC-5 strain was also able to produce GlcNAc monomers on TLC plates. The chitinase of PRC-5 inhibited the mycelial growth of Rhizoctonia solani KACC40111, which indicates that it could be used as a biocontrol agent for phytophathogens. The chitinase isozyme N1, which had a molecular weight of 62 kDa, was transferred from a native and SDS-PAGE gel onto an immunoblot and was probed using an anti-PrGV-chitinase.

Expression patterns of chitinase and chitosanase produced from Bacillus cereus in suppression of phytopathogen.

Bacillus cereus MP-310 was incubated on various culture media substrates as LB, colloidal chitin, chitosan powder, and chitosan beads to investigate the concurrent expression patterns of chitinase and chitosanase isozymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chitinase activity increased rapidly with a maximum level after 6 days of incubation in CM-chitin medium. Major bands of chitinase isozymes were strongly expressed on SDS-PAGE in LB medium (four bands) and in colloidal chitin medium (five bands) after 6 days after incubation, and in chitosan powder medium (one band) and in chitosan beads medium (five bands) after 12 days after incubation. A major band of chitosanase isozymes was strongly expressed on SDS-PAGE in chitosan powder medium (one band) and in chitosan beads medium (one band) after 12 days of incubation.

Nematode-antagonistic effects of Cinnamomum aromaticum extracts and a purified compound against Meloidogyne incognita

The potential use of Cinnamomum aromaticum and its active compound to control Meloidogyne incognita was investigated in vitro and in pot experiments. One compound, cinnamyl acetate, was isolated by thin layer chromatography and silica gel column chromatography, and identified by 1H-NMR, 13C-NMR and mass spectrometry. Juvenile movement and hatch inhibition by cinnamyl acetate was dependent on both the concentration and incubation time of the cinnamyl acetate. Treatment with 25, 50, 75, 100 and 125 μg ml−1 of cinnamyl acetate resulted in 33.7, 65.1, 81.1, 100 and 100% inhibition of movement of second-stage juveniles, respectively, at 50 min after incubation. The juvenile movement inhibition was <20% at the tested concentrations at 10 min after incubation. Cinnamyl acetate treatment resulted in 20.8, 39.4, 81.3 and 90.7% hatch inhibition at 25, 50, 100 and 200 μg ml−1, respectively, at 3 days after incubation and 21.6, 39.3, 73.2 and 88.7% hatch inhibition at 25, 50, 100 and 200 μg ml−1, respectively at 6 days after incubation. In pot tests, C. aromaticum crude extracts effectively inhibited infection of M. incognita on cucumber plants. Cinnamomum aromaticum crude extracts applied at 0.5 and 1.0 mg (g soil)−1 significantly reduced the numbers of galls caused by M. incognita. The activities of pathogenesis-related proteins as β-1,3-glucanase and peroxidase on leaves of plants treated with C. aromaticum crude extracts were significantly higher than those on leaves of control plants.

Expression and degradation patterns of chitinase purified from Xuehuali (Pyrus bretschneiderilia) pollen

The present study investigated the expression pattern of chitinase in Xuehuali (Pyrus bretschneiderilia) pollen, as well as its subsequent degradation. The chitinase was purified and collected using chitin affinity column chromatography with regenerated chitin. After purification, four additional chitinase isozymes (chiA, chiB, chiC, and chiD) and chitinase (Chi II) were clearly expressed on SDS-PAGE gels that contained 0.01% glycol chitin. The chitinase reaction products were examined using GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6 as substrates at 2 and 24h after reaction via TLC and HPLC. The (GlcNAc)4 oligosaccharide was slightly degraded to (GlcNAc)2 after 24h of reaction with Xuehuali pollen chitinase on TLC. Meanwhile, (GlcNAc)5 was degraded to (GlcNAc)2-4, and 2300ppm (GlcNAc)6 was degraded to 246ppm (GlcNAc)2, 208ppm (GlcNAc)3, 572ppm (GlcNAc)4, and 336ppm (GlcNAc)5 on HPLC. With regard to temperature, the strongest Xuehuali pollen chitinase activity (0.69 unit/mL) was observed at 37°C after 3h of incubation, and with regard to pH, the strongest activity (0.72unit/mL) was observed at pH 3 after 3h of incubation. The main chitin oligomers degraded from (GlcNAc)6 were (GlcNAc)2 and (GlcNAc)4.